The aim of this proposal is to learn more about the tissues that influence the last stages of sympathetic neuron differentiation. My hypothesis is that the embryonic connective tissues that develop in the midst of primary sympathoblasts influence them differentiate into two distinct types of neurons, namely, the type II small intensely fluorescent (SIF) cell or paraneuron, and the principal neuron (PN). Adult PN are the predominant type of neuron in paravertebral sympathetic ganglia which develop near the vertebral column, whereas adult SIF cells are the predominant cell type in prevertebral sympathetic ganglia which develop near the dorsal aorta. To test the hypothesis two kinds of experiment will be carried out: 1) neural crest will be combined with embryonic connective tissues from two different regions and grafted to the chorioallantoic membrane (CAM) of chick hosts. 2) Neural crest will be cultured on substrates of the same two connective tissues. The neural crest cells will be marked with the quail nucleolar marker so they can be distinguished from chick cells. The embryonic connective tissues will be dissected from the region of the developing vertebral column and the dorsal aorta. The patterns of specific neuronal differentiation will be assessed with the formaldehyde induced fluorescence technique and electron microscopy. If significantly more PN differentiate when neural crest is combined with vertebral connective tissue compared with dorsal aorta connective tissue, and vice versa for SIF cells, then this result will indicate that tissue factors influence sympathetic neuronal differentiation. Controls will check for 1) self-differentiation of precursor cells in the absence of embryonic connective tissues, and 2) differentiation and maintenance of sympathetic neurons under the experimental conditions. Additional experiments will explore the relationship among homonal, cellular, and extracellular matrix influences on differentiation. The results will lead to further studies determining the nature of the factors involved. The results will be important for understanding how functional differences appear in a class of neurons that play important roles in human behavior and autonomic physiology. If the tissue environment does influence sympathetic differentiation, then this information could provide the basis for new therapy in cases where autonomic neurons fail to appear normally.